Spin trapping compounds

ABSTRACT

Compositions containing as the active ingredient a spin-trapping reagent, preferably α-phenyl butyl nitrone (PBN) or spin-trapping derivatives thereof, in a suitable pharmaceutical carrier for administration to a patient are disclosed for treating or preventing symptoms associated with aging or other conditions associated with oxidative tissue damage. Other spin-trapping agents can also be used, such as 5,5-dimethyl pyrroline N-oxide (DMPO) or α-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), and other spin-trapping derivatives thereof. These compositions and methods are useful in the treatment of age-related disorders, pre-surgical and/or pre-anesthetic preparation or administration of chemotherapeutic agents, and in the treatment of disorders or trauma of the brain, cardiovascular system, and lymphatic system. Studies in animals demonstrate that administration of compound for a two week period reduces the level of oxidized brain enzymes to normal and restores memory to the same level as tested in young control animals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/365,548, filed Dec.28, 1994, now U.S. Pat. No. 5,578,617, which is a divisional ofapplication Ser. No. 08/027,559, filed Mar. 5, 1993, now U.S. Pat. No.5,405,874, which is a continuation of application Ser. No. 07/589,177,filed Sep. 27, 1990, now abandoned, which is a continuation-in-part ofapplication Ser. No. 07/422,651, filed Oct. 17, 1989, now U.S. Pat. No.5,025,032.

BACKGROUND OF THE INVENTION

The present invention is a method and compositions containing spintrapping agents for the treatment of age related dysfunctions and otherconditions arising from oxidative damage.

Age related changes in central nervous system function have generallybeen associated with the loss of cells, a widening of lateral ventriclesand deficits in short term memory. The precise mechanisms of functionalchanges as a result of aging, or other diseases associated with aging,have not generally been agreed upon.

Several mechanisms for the generation of oxidized material in the brainhave been proposed. In particular, transition metals, especially ironand copper, have been suggested as mediating aspects of this oxidation.A marked reduction in certain neurotransmitter receptor systems has beenassociated with increased oxidation of proteins. For example, decreasesin muscarinic receptors and other cholinergic systems have beencharacterized as they relate to alterations in functions in Alzheimersdisease. It has also been hypothesized that aging is associated withmultiple minor periods of ischemia (multi-infarct conditions ortransient ischemia attacks) which, over a period of time, may give riseto the production of oxidized protein.

Changes associated with ischemic brain disease have been proposed to bethe result of alterations in calcium disposition, increase in excitoxicneurotransmitter release, production of free radicals and the attendantacidosis that results in an increase in the loosely related metals inthe cell that are catalytic for the generation of oxygen free radicals.These changes are largely limited to neuronal elements. Reactive gliahave been demonstrated, however, they are mostly associated withpostneuronal damage.

The treatment of age related dementias have been largely limited by theinability to develop an appropriate model for the study of thiscondition. This is due to the fact that aging is a very complicatedcondition which is difficult to model, especially with the lack ofspecific information associated with the functional and biochemicalbasis of human age related dementias. The use of animal models haslargely depended upon model systems used in brain studies, where thebrains are not truly senescent, or the use of senescent animals, withlittle understanding of the origin of the senescence or, in some cases,the inability to demonstrate truly functional senescence.

The demonstration in a variety of systems, both neural and nonneural,that there is an age related enhancement of the level of oxidizedprotein in tissue gives rise to the possibility that age relateddysfunctions in the central nervous system may be associated with thebuild-up of oxidized proteins and oxidized macromolecules within neuronsthroughout the central nervous system. The hypothesis is that cellswhich have a buildup of oxidized protein are less functional and lessable to maintain the specified role of those cells in that particulararea of the central nervous system. While this hypothesis has beensuggested by several investigators, there are no reports of substantialinvestigations in which alterations in the oxidized protein burden ofthe central nervous system was manipulated and correlated with afunctional outcome on the part of the animal. Such an approach, if trulyassociated with brain dysfunction, would provide a basis for reversingthe age related neuronal deficit of cells that are still viable. Thus,such an approach is targeted at cells which are marginally functionalbut still viable.

It is therefore the object of the present invention to providecomposition and methods for the use in preventing or reversing agerelated functional deficits.

It is further the object of the present invention to provide compositionand methods for use thereof which are useful in preventing and reversingcognitive deficits associated with infection or inflammation.

It is another object of the present invention to provide composition andmethods reducing post traumatic cognitive dysfunction.

SUMMARY OF THE INVENTION

Compositions containing as the active ingredient a spin-trappingreagent, preferably α-phenyl butyl nitrone (PBN), or spin-trappingderivatives thereof, in a suitable pharmaceutical carrier for patient,are disclosed for treating or preventing symptoms associated with agingor other conditions associated with oxidative tissue damage. Thepreferred PBN compositions have the following general formula: ##STR1##wherein: X is phenyl or ##STR2## wherein R is H, ##STR3## and n is awhole integer from 1 to 5; or ##STR4## Y is a tert-butyl group that canbe hydroxylated or acetylated on one or more positions; phenyl; or##STR5## wherein W is ##STR6## and Z is a C₁ to C₅ straight or branchedalkyl group.

Other spin-trapping agents can also be used, such as 5,5-dimethylpyrroline N-oxide-(DMPO)-or α-(4-pyridyl 1-oxide)-N-tert-butylnitrone(POBN), and other spin-trapping derivatives thereof.

In the preferred embodiment, the compositions are administered one totwo times daily by oral administration, at a dosage equivalent tobetween one and ten milligrams PBN/70 kg of human body weight. Studiesin animals demonstrate that administration of compound for a two weekperiod reduces the level of oxidized brain enzymes to normal andrestores memory to the same level as tested in young control animals. Asignificant reduction in oxidized proteins and memory recovery isobserved as early as seven days after initiation of treatment; levelsare still comparable to young controls one to three days followingcessation of treatment, and partially reduced at seven days followingcessation of treatment.

These compositions and methods are useful in the treatment ofage-related disorders, pro-surgical and/or pro-anesthetic preparation oradministration of chemotherapeutic agents, and in treatment of disordersor trauma of the brain, cardiovascular system, lymphatic system, and,potentially, in the treatment of some viral disorders characterized byoxidation of host proteins in cells infected by the virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the alkaline protease activity from gerbil cortex(% of young, three to four month old gerbil cortex) for young gerbils(age three to four months), old gerbils (retired breeders of twelve tofifteen months of age), old gerbils that received twice daily injectionsof 0.1 ml saline/kg body weight, and old gerbils that received twicedaily injections of 10 mg PBN in saline/kg body weight for two weeks.Protease activity was determined using oxidized protein extracted fromyoung gerbil cerebral cortex.

FIG. 2A and FIG. 2B are graphs comparing protein carbonyl activity(pmol/mg protein) (FIG. 2A) and glutamine synthetase activity (FIG. 2B)in the cerebral cortex (neocortex) of young adult and old gerbils overdays of administration of 32 mg PBN/kg administered twice a day for one,three, seven or fourteen days. At the end of each of the days indicated,animals were decapitated and cerebral cortex removed and rapidly frozenin liquid nitrogen. Protein carbonyl content was determined using theDNPH procedure. The results demonstrate the reduction in oxidativedamage to proteins and the loss of enzyme activity in gerbil cerebralcortex as a result of twice daily administration of 32 mg PBN/kg (i.p.).Each histogram is the mean of three subjects.

FIG. 3A, 3B and 3C are graphs comparing changes in protein carbonyl(pnol/mg protein) (FIG. 3A), glutamine synthetase (FIG. 3B), andprotease activity (% control) (FIG. 3C) over days following terminationof twice daily dosing with 10 mg PBN/kg body weight. FIG. 3A representsthe level of carbonyl in the soluble protein obtained from gerbilstreated for fourteen days and tested at 1, 3, 7 and 14 days post dosing.FIG. 3B is the time related decrease in cortical glutamine synthetase(GS) activity after termination of twice daily injections of PBN. FIG.3C demonstrates the time related decrease in alkaline protease activityfollowing termination of twice daily injections of PBN. Each histogramis the mean±standard error (S.E.) of three subjects at each of theindicated times. The asterisk and dashed line indicates the old gerbil,untreated control values for each of the measures.

FIG. 4A, 4B and 4C are graphs comparing changes in protein carbonyl(pnol/mg protein) (FIG. 4A), glutamine synthetase (FIG. 4B), andprotease activity (% control) (FIG. 4C) over days following terminationof twice daily dosing with 32 mg PBN/kg body weight. FIG. 4A representsthe level of carbonyl in the soluble protein obtained from gerbilstreated for fourteen days and tested at 1, 3, 7 and 14 days post dosing.FIG. 4B is the time related decrease in cortical glutamine synthetase(GS) activity after termination of twice daily injections of PBN. FIG.4C demonstrates the time related decrease in alkaline protease activityfollowing termination of twice daily injections of PBN. Each histogramis the mean±S.E. of three subjects at each of the indicated times. Theasterisk and dashed line indicates the old gerbil, untreated controlvalues for each of the measures.

FIG. 5 is a graph of the eight arm radial arm maze performance of youngor old gerbils treated with either saline or PBN. Gerbils were placedinto the central compartment of the maze with the barrier in place tolimit exploration. After the barrier was removed, the number of armsre-entered and the total elapsed time before all eight arms were enteredwas recorded. Each histogram represents the mean±of 18 gerbils. Theanimals were administered PBN twice daily (either 10 or 32 mg PBN/kgbody weight) for seven days and tested at the end of seven days ofdosing.

DETAILED DESCRIPTION OF THE INVENTION

It has now been discovered that, further to the methods using PBN forthe treatment and prevention of ischemic damage described and claimed inU.S. Ser. No. 07/422,651 filed Oct. 17, 1989, now Reissue U.S. Pat. No.35,112, spin-trapping agents are useful in preventing or treatingsymptoms associated with aging, trauma, drug administration and surgery,especially of the brain. As used herein, a free radical scavenger orspin-trap reagent is a molecule that will form a stable complex withfree radical. A free radical carbon trap is a molecule in which the freeradical is localized on a carbon atom or a nitrogen atom. As a result ofthis chemical bond formation, the free radical is no longer damaging tothe cell. In combination with a pharmaceutical vehicle suitable foradministration to a patient, preferably by oral administration, thesecompounds are useful in preventing or reversing symptoms associated withaging, for example, increased levels of oxidized proteins, decreasedenzymatic activity, and spatial and short term memory. Currently, thereare no effective, non-toxic treatments for aging. Effectiveness has beendemonstrated in animals after as few as seven days of administration.Effectiveness continues for at least one week after administration.Values return to pretreatment levels after two weeks.

Useful Spin-trapping Compounds

PBN and Derivatives Thereof

The preferred spin-trapping compounds are α-phenyl t-butyl nitrone(PBN), and derivatives thereof. PBN has no measurable effect on normalor uninjured cells. PBN is the preferred compound at this time, althougha number of derivatives are also useful, including hydroxy derivatives,especially 2-, 3- or 4-hydroxy PBN and mono-, di- and trihydroxytert-butyl nitrone; esters, especially esters which release 2-, 3, or4-hydroxyphenyl t-butyl nitrone such as the acetoxy derivative, 2-, 3-,or 4-carboxyphenyl t-butyl nitrone, such as the ethyl derivative, orphenyl hydroxybutyl nitrone, such as the acetoxy derivative; alkoxylderivatives, especially alkoxyl derivatives which release 2-, or4-hydroxyphenyl t-butyl nitrone, such as the methyl derivative; andacetamide derivatives, especially acetamide derivatives which release2-, or 4 aminophenyl t-butyl nitrone, such as the acetyl derivative;diphenyl nitrone (PPN) and the analogous diphenyl nitrone derivatives.As used herein, "PBN" refers to both phenyl t-butyl nitrone andderivatives thereof, unless otherwise stated.

The general formula for PBN and useful derivatives thereof is: ##STR7##wherein: X is phenyl or ##STR8## wherein R is H ##STR9## and n is awhole integer from 1 to 5; or ##STR10## Y is a tert-butyl group that canbe hydroxylated or acetylated on one or more positions; phenyl; or##STR11## wherein ##STR12## and Z is a C₁ to C₅ straight or branchedalkyl group.

Other Spin-trapping Reagents

Other spin-trapping agents can also be used, such as 5,5-dimethylpyrroline N-oxide (DMPO) or α-(4-pyridyl 1-oxide)-N-tert-butylnitrone(POBN), and spin-trapping derivatives thereof. Derivatives are madeusing standard techniques, for example, for substitution of the methylgroups. The general formula for DMPO is: ##STR13## wherein A and B areindependently CH₃, CH₂ OH, CH₂ OW, or ##STR14## n is an integer from 1to 5 wherein W is ##STR15## and Z is a C₁ to C₅ straight or branchedalkyl group.

The general formula for POBN is: ##STR16## wherein Y is a tert-butylgroup that can be hydroxylated or acetylated on one or more positions;phenyl; or ##STR17## wherein W is ##STR18## and S=H, (OR)_(n), wherein Ris H, ##STR19## n is a whole number from 1 to 4, or ##STR20## Z is a C₁to C₅ straight or branched alkyl group.

Indications That the Compositions are Useful in Treating

The free-radical scavenger compositions are useful in treating a varietyof dysfunctions or disorders characterized by oxidized proteins in thetissues or cells. Oxidation of cytosolic protein has been demonstratedto occur in a wide variety of pathological conditions. Accordingly,compounds which have as their fundamental mechanism of action theinterference of production of oxidized protein should be useful in thetreatment of a wide variety of diseases having what appears at firstglance to be widely dissimilar etiologies, because the fundamental causeof the condition is oxidation of protein or nucleic acids.

In one embodiment, the spin-trapping agent is administered to a patientto reverse the damage occurring as a function of age. Preliminaryresults indicate that there is a net increase in the oxidation ofproteins and the accumulation of oxidized material in the brain. Thedevelopment of senil plaque is also routinely observed in aged patients.

Other disorders are those resulting from trauma, such as a blow to thehead, or from drug treatment, for example, administration of anesthesiaor drug abuse, or even as a result of some types of viral infections.

It has now been determined that the level of oxidized brain proteinappears to be inversely related to performance in a short term memorytask and directly related to risk of stroke-induced damage andbehavioral change. Increased cellular oxidation may result in one ormore of the following: (a) oxidative damage to cellular proteins couldcause a change in the regulation of ion channels, there could be achange in the rate and efficiency of signal translation and membranedepolarization, significant changes in energy fluxes may occur andcompromise selective function, the fidelity of RNA transcription may bealtered due to oxidative damage to DNA, RNA translation may be affectedeither by oxidation of the RNA or regulatory macromolecules, or the rateof protein degradation may be altered.

Any of these changes could negatively impact on the acquisitionconsolidation and retrieval of information, even by interference with asingle step in the learning and memory process. It is possible thatoxidation of cells in a particular brain region could result inacquisition deficits, whereas oxidation of a different region couldresult in output deficiency. Considering the number of devastatingneurodegenerative diseases, including Alzheimer's disease, thistreatment could potentially be a tremendous help to people with thesedisorders.

Examples of other disorders that can be treated with these compositionsinclude peripheral neuropathy of diabetes, exercise induced muscledamage and pain, and enhancement of cellular response to hormonalsignals.

Treatment of Neurodegenerative Disorders

Several neurodegenerative conditions are most appropriately treated bycompounds that interfere with protein oxidation. Alzheimer's disease hasbeen associated with the accumulation of abnormal oxidized proteins orthe production of abnormal proteins in areas that are pathologicallyaffected. In addition, age related enhancement in protein oxidationoccurs in all cells in the aged individual. PBN and derivatives thereofhave been demonstrated to be useful in the reduction in proteinoxidation and in the increase in the activity of critical enzymes withinthe brain of aged animals. Since this is a fundamental change inoxidative state, it is likely that PBN and other related compounds wouldbe useful when given chronically to individuals who are in the earlyphases, or possibly in the late phases, of Alzheimer's disease. Inaddition, multi-infarct dementias should be treatable with thesecompounds, since they also deal with ischemia reperfusion oxidationissues.

Senile dementia has not been directly evaluated for ischemia reperfusionetiology or protein oxidation, however, it is likely that seniledementia would also be treatable with these compounds. This is based onthe hypothesis that advanced age is associated with increased productionof oxidized protein. In progeria, a unique condition in which aging isaccelerated, Stadtman and colleagues at the NIH have demonstrated thatthere is a marked increase in the base-level of oxidized protein even inyoung adult subjects with progeria, as reported by Oliver, et al., J.Biol. Chem. 262, 5488-5491 (1987) and Starke-Reed and Oliver, Arch.Biochem. Biophys. 275, 559-567 (1989). While this is a rare condition itshould also be treatable with these compositions.

Another condition which is likely to be associated with oxidative damagearising from microcirculatory difficulties is the diabetic peripheralneuropathies and vascular change. These are tragic conditions in whichamputation is eventually necessary in order to save the patient. Whilethese compounds are not likely to improve vascular flow they are likelyto reduce the impact of transient changes in vascular flow which resultin oxidation and damage to the peripheral nerves and also in damage tothe skeletal muscle which is often associated with the condition calledexercise induced or intermittent claudication. If the retinopathyassociated with diabetes is also an ischemia reperfusionmicrocirculation problem, then the spin-trapping compounds will beuseful in treating the retinal damage which occurs very frequently indiabetic patients.

Pre-surgical Preparation

Since the status of the cell and its survival in a hypoxic or anoxicenvironment is dependent upon the ability of the cell tocompartmentalize metals and handle oxygen in a useful manner, incontrast to peroxidation, it is expected that these compounds will beuseful as presurgical preparatory medication to reduce the carbonyl loadand improve the enzyme status of the patient prior to elective surgery.These compounds would also help the cells of the body achieve a higherlevel of enzymatic function, shorten the recuperative phase, and reducethe likelihood of any interoperative complications associated withchanges in microcirculation.

Treatment of Viral Infections and Inflammatory Disorders

Retroviruses selectively infect certain types of cells, such aslymphocytes. An example of a retrovirus that has been the subject ofmuch research activity is the human immunodeficiency virus (HIV), whichcauses Acquired Immunodeficiency Syndrome (AIDS). No means forprevention of infection has been found, although there have beennumerous attempts to find a treatment. Since activation of lymphocytesis associated with the oxidation of protein and activation oflymphocytes is required prior to release of newly formed viruses, it isexpected that administration of these compositions will inhibitinfection and replication of lymphocytes by the viruses. The activationof the T-4 lymphocyte is associated with a cascade of biochemicalintracellular changes, one of which is the production of oxidizedprotein. If PBN related compounds can block protein oxidation in theabnormal process then it is possible that PBN or other spin-trappincompounds could in fact interfere with the process of viral replicationand/or dissemination of the virus from the host cell (T-4 lymphocyte),thereby acting as a virustatic agent by preventing the T-4 lymphocytefrom releasing the newly formed viruses. This would be analogous to theuse of isoniazid (INH) in the treatment of tuberculosis. At low dosesINH is a tubercula static in that it reduced the infectivity and spreadof the tuberculosis, thereby effectively protecting the patient frompulmonary damage.

It is important to note that the effects of the spin-trapping compoundsoccur in animals that have a base-level of carbonyl formation whichappears to be necessary for post translational processes. Old animalshave a significantly elevated level of carbonyl protein which isassociated with decreased enzymatic function relative to young controlanimals. When young control animals are given the exact same dosageregimen, there is no significant change in enzyme activity nor is theresignificant change in protein carbonyl. Thus the PBN and relatedspin-trapping compounds are not likely to interfere with fundamentalprocesses that are necessary for the normal cellular function.

In conclusion, a number of clinical conditions appear to have as theirfundamental cause oxidation of cellular protein and enzymatic damage.Spin-trapping compounds are effective in animal models in reducing theprotein oxidation and improving enzymatic function. This occurs inpreparations in which the abnormal oxidized protein is modified andprotected but the normal post translational oxidation is allowed tooccur. This would suggest that PBN does not interfere with the normalnecessary oxidation of proteins following synthesis.

Effective Dosages of PBN

Exemplary dosages of PBN range from 0.1 to 10 mg/kg of body weight inanimals. The effective dosage of PBN in humans is expected to be betweenapproximately 1 and 10 mg/70 kg body weight. Toxicity tests havedemonstrated that the compound is completely innocuous, with such lowtoxicity that it was not possible to determine an LD₅₀.

In the preferred application, the PBN is administered to a patientsuffering from memory loss or other symptoms frequently associated withaging. Optimum results are generally observed after two weeks of dailyor twice daily oral administration. The compositions can also beeffectively administered prior to, during or shortly after surgery, andprevent or decrease the extent of cellular damage resulting from eitherthe trauma or anesthesia.

Since the trapping of endogenous free radicals is specific for onlythose cells that have been exposed to the conditions that result in theproduction of free radicals, the traps have little or no effect onnormal cells. The beneficial effects occur only in injured cells, and donot require the presence of specific receptors, specific enzymes, and/orspecific cell types.

Methods of Administration of PBN

The PBN is preferably administered systemically, most preferably orally,since this is the most rapid and efficient means for delivering theactive compound to the site of free radical generation. The PBN may beadministered at once, or may be divided into a number of smaller dosesto be administered at varying intervals of time. Other methods ofadministration can also be used, including subcutaneous, intravenous,and intraperitoneal administration. The concentration of active compoundin the drug composition Will depend on absorption, inactivation, andexcretion rates of the drug as well as other factors known to thoseskilled in the art. The effective dosage may also be determined based onthat amount required to prevent or reverse predisposition of the cellsto damage resulting from depletion of ATP (as demonstrated by in vivoNMR) and damage from free radical generation. It is to be noted thatdosage values will also vary with the condition of the patient beingtreated. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedcomposition.

A preferred mode of administration of the active compound is in a formfor oral delivery. Oral compositions will generally include an inertdiluent or an edible carrier. Preferred pharmaceutical carriers forintravenous administration are saline or phosphate buffered saline atphysiological pH. Since PBN degrades at pH less than approximately 3 to4, it is preferred to administer the PBN at a pH of 4 or higher, or incombination with food, a buffering agent, or in an enteric coating. Fororal delivery, the PBN may be enclosed in capsules, compressed intotablets, microencapsulated, entrapped in liposomes, in solution orsuspension, alone or in combination with a substrate immobilizingmaterial such as starch or poorly absorbable salts such as immodium.Pharmaceutically compatible binding agents can be included as part ofthe composition. The tablets or capsules may contain, for example, anyof the following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel®, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier. In addition, dosage unit forms can containvarious other materials which modify the physical form of the dosageunit, for example, coatings of sugar, shellac, or other enteric agents.

The present invention will be further understood with reference to thefollowing non-limiting examples demonstrating methods for determiningeffectiveness of PBN administration for treatment or prevention and/orreversal of symptoms associated with aging.

Example 1

Determination of Brain Enzyme Levels in Old Versus Young Gerbils Treatedwith PBN

A correlations between the duration of ischemia and either the change inspontaneous behavior or the level of oxidized brain protein haspreviously been demonstrated in gerbils. Many other psychiatric andneurological conditions have been proposed to be the result ofoxidation. Among these conditions, cellular aging has been associatedwith oxygen radicals and the accumulation of proteins. The level ofoxidized protein, glutamine synthetase activity, brain protease activityand radial arm maze performance in young adult and retired breedergerbils have now been compared and demonstrate that there is a directrelationship between the age of the subject and the level of oxidizedbrain protein, as measured using a protein carbonyl assay. Increasedlevels of protein carbonyl were associated with decreased glutaminesynthetase activity and decreased alkaline protease activity. Incontrast, there was no change in acid protease activity of retiredbreeders, compared to young adult gerbils. Consistent with theage-related increase in protein oxidation and enzyme damage, retiredgerbils made significantly greater numbers of errors in a test ofshort-term memory, compared to young adult gerbils. These studiesdemonstrate that the functional deficits that occur as a result of agingmay be associated with increased protein oxidation and decreased brainenzyme activities.

This system has been used to demonstrate the effectiveness of PBN inrestoring young brain enzyme levels and short term memory to oldanimals. The results are shown in FIGS. 1 through 5, as follows. Younggerbils were obtained from Tumblebrook Farms, West Brookfield, Mass.,weighing 50-60 grams and age three to four months. Control gerbils weregiven saline. Animals were killed by decapitation and their brainsremoved for analysis.

FIG. 1A is a graph of the percent alkaline protease in young (three tofour month old) gerbils, old (twelve to fifteen month old retiredbreeder) gerbils, old gerbils administered 0.1 ml saline twice daily(b.i.d.), and old gerbils administered 10 mg PBN/kg body weight b.i.d.for fourteen days.

The results demonstrate that PBN is effective in restoring alkalineprotease levels in old animals to those levels present in young animals.

FIG. 2A is a graph of the changes in protein oxidation from brains ofyoung and senescent gerbils, plotting nmol protein carbonyl/mg proteinversus days of treatment with PBN. The gerbils were given twice dailyinjections of 32 mg/kg PBN for fourteen days. Animals were killed atone, three, seven and fourteen days and protein carbonyl levelsdetermined.

As can be seen in the figure, there is no change in the level ofoxidized protein of young gerbils treated for up to 14 days with PBN.This indicates that the level of oxidized protein is likely to be anatural and necessary post translational effect, for example, if aftersynthesis of the protein, the protein is activated by a modificationinvolving carbonyl oxidation. In contrast to the carbonyl level seen inyoung animals, control aged animals (15 months of age) have a markedincrease of carbonyl content. This increased carbonyl content isresponsive to treatment with PBN. Multiple days of treatment with PBNresults in a progressive reduction in the level of protein carbonyl tothe level seen in young animals.

The level of protein carbonyl reduction (oxidized protein burden ofneurons in the brain) is only to the level of the normal young gerbilbrain. Neither the levels in the young gerbil brain nor the levels inthe senescent gerbil brain can be further reduced beyond this level.This observation supports the hypothesis that there is a necessary levelof oxidation that occurs in cells in normal animals, which is requiredfor cells to have "normal function", and that control aged animals (15months of age) have a marked increase of carbonyl content. Thisincreased carbonyl content is responsive to treatment with PBN. Theability of PBN to reduce the protein carbonyl load of cells alsoindicates that this is an active oxidation process which occurs at aregular or predictable rate and that there are mechanisms existentwithin the cells of the brain which can remove this oxidized protein ifthe process is interrupted.

FIG. 2B compares the levels of glutamine synthetase (gs) in young andold animals and evaluates the effects of daily administration of PBN onthe specific activity of the enzyme. This particular enzymatic markerhas been selected because it is a highly sensitive protein to oxidationand because it is a metalloprotein that has bound to it metal which mayparticipate in the generation of free radicals if the metal isdissociated with its binding site. Glutamine synthetase activity hasbeen used by Stadman and colleagues (Oliver, et al., Proc. Natl. Acad.Scie. U.S.A. 87, 5144-5147 (July 1990)) as a marker enzyme foralterations following protein oxidation.

As shown in FIG. 2B, the level of glutamine synthetase is lower in oldgerbils (1.2) than in young adult gerbils (2.1). This is consistent withprevious studies in which increases in the level of the carbonyl protein(oxidized protein in cells) is associated with a decrease in glutaminesynthetase activity. In particular, if the glutamine synthetase enzymeis purified and the carbonyl content of that enzyme is evaluated, thereis a marked increase in the level of oxidized protein in the presence oflowered glutamine synthetase activity. As also demonstrated in FIG. 2B,repeated administration of PBN in young gerbils had no effect onglutamine synthetase activity, providing further evidence that the levelof carbonyl is associated with normal function and chronicadministration of PBN has no effect on either the level of carbonyl oron the marker enzyme activity. In contrast to the young gerbils, oldgerbils given daily injections of PBN show a time related increase inglutamine synthetase activity that parallels the reduction in proteincarbonyl content, indicating that the reduction in oxidized proteinburden of cells is associated with a recovery of the enzymatic activityto the normal level seen in young adult gerbils.

It is important to note that there is no increase in enzymatic activityabove that seen in young adult gerbils. Thus the treatment with PBNreverses the effect of aging on enzymatic activity but does not resultin an activation of enzyme activity that exceeds the normal values.

Example 2

Determination of Residual Effect of PBN on Reduction of Brain EnzymeLevels

As shown in FIGS. 3A, 3B, and 3C, as compared with FIGS. 4A, 4B, and 4C,administration of 10 mg PBN/kg is as effective as administration of 32mg PBn/kg body weight in restoring young enzyme levels. The appropriatedosages and full range of effective dosages for other species of animalscan be determined using a similar methodology.

The time related changes in protein oxidation and enzyme activityfollowing termination of twice daily dosing with either 10 mg PBN/kg(FIGS. 3A, B, and C) or 32 mg PBN/kg (FIGS. 4A, B, and C) are also shownby these figures. The results demonstrate that the effect of the PBN isunaltered one to three days after termination of treatment twice dailywith the PBN, although the PBN itself has a half-life of three hours. Atseven days, the enzyme levels are altered by approximately 50%. Atfourteen days, the oxidized enzymes have returned to approximately theirpre-treatment levels.

Example 3

Demonstration of Correlation Between Effect of PBN on Brain Enzymes andMemory

FIG. 5 demonstrates that there is a functional counterpart to suchtreatment. Young and old gerbils were tested in a radial arm maze testfor spacial and short term memory. Animals were placed in the centralhub of a eight armed radial maze and given access to explore all eightarms of the radial arm maze. When the animal completed the test ofexploring each of the eight arms, the animal was removed from the maze.The number of times that the animals reentered arms that had beenpreviously entered was counted as an error. Under ideal conditionsanimals will enter each one of the eight arms but not reenter any of thearms. In many cases young adult gerbils (young) entered each of the armswithout reentering any of the arms. The time required to enter all eightarms was also recorded but did not appear to determine the efficiency ofthe short term memory task. As can be seen in FIG. 5, young gerbils madean average of 2.83 errors during the test session. In contrast, oldgerbils made an average of 6.82 errors, which is a highly significantdifference between the two groups.

Young adult gerbils exposed to a period of transient ischemia, which isalso an oxidizing process, make a substantial number of errors with anaverage value of 15 errors in such a test session. During the postischemic period, there is a marked build up of carbonyl protein andreduction in glutamine synthetase activity similar to that which is seenin aging.

Treatment of gerbils for seven days results in a marked alteration inthe number of errors seen with old gerbils. Young gerbils given PBN forseven days were not significantly different from control gerbils whereasthe old gerbils given PBN for seven days showed a marked reduction inthe number of errors and returned to the range of performance seen withcontrol young gerbils. Thus, there is a functional counterpart to thebiochemical changes that are seen in that reduction in protein carbonyland the increase in glutamine synthetase activity following chronic PBNtreatment. This functional counterpart is demonstrated by the number oferrors seen in a short term spacial memory task, the radial arm maze. Itshould be noted that the radial arm maze test does not require any foodreinforcement or any other reward associated with the test. Naiveanimals are placed into the radial arm maze, are tested once and thesedifferences are reliable on retest.

Modifications and variations of the method and composition for thetreatment of aging will be obvious to those skilled in the art from theforegoing detailed description. Such modifications and variations areintended to come within the scope of the appended claims.

We claim:
 1. A compound of the formula: ##STR21## Y is a tert-butylgroup that can be hydroxylated or acetylated on one or more positions;phenyl; or ##STR22## and wherein Z is a C₁ to C₅ straight or branchedalkyl group.
 2. The compound of claim 1 wherein X is ##STR23##
 3. Thecompound of claim 2 wherein Z is methyl.
 4. The compound of claim 3wherein Y is a tert-butyl group.
 5. The compound of claim 1 wherein X is##STR24##
 6. The compound of claim 1 wherein Y is a tert-butyl groupthat can be hydroxylated or acetylated.
 7. The compound of claim 1wherein Y is phenyl.
 8. The compound of claim 1 wherein Y is ##STR25##9. A compound of the formula: ##STR26##
 10. A compound of the formula:##STR27## wherein: X is ##STR28## wherein R is H, ##STR29## and n is awhole integer from 1 to 5; or ##STR30## Y is a tert-butyl group that canbe hydroxylated or acetylated on one or more positions; or ##STR31## andwherein Z is a C₁ to C₅ straight or branched alkyl group.
 11. Thecompound of claim 10 wherein X is ##STR32##
 12. The compound of claim 11wherein Z is methyl.
 13. The compound of claim 10 wherein X is ##STR33##14. The compound of claim 10 wherein X is ##STR34##
 15. The compound ofclaim 10 wherein Y is a tert-butyl group that can be hydroxylated oracetylated.
 16. The compound of claim 10 wherein Y is ##STR35##
 17. Acompound of the formula: ##STR36## wherein Y is a tert-butyl group thatcan be hydroxylated or acetylated on one or more positions; phenyl; or##STR37## and S=(OR)_(n), wherein R is H, ##STR38## Y is as definedabove, n is a whole number from 1 to 4, or ##STR39## and Z is a C₁ to C₅straight or branched alkyl group.
 18. The compound of claim 17 wherein Yis a tert-butyl group that can be hydroxylated or acetylated.
 19. Thecompound of claim 17 wherein Y is phenyl.
 20. The compound of claim 17wherein Y is ##STR40##
 21. A compound of the formula: ##STR41## whereinA and B are independently CH₂ OW, or ##STR42## and n is an integer from1 to 5,wherein W is ##STR43## and Z is a C₁ to C₅ straight or branchedalkyl group.
 22. The compound of claim 21 wherein at least one of A andB is ##STR44##
 23. The compound of claim 21 wherein at least one of Aand B is CH₂ OW.